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Sample Preparation and Extraction in Small Sample Volumes Suitable for Pediatric Clinical Studies: Challenges, Advances, and Experiences of a Bioanalytical HPLC-MS/MS Method Validation Using Enalapril and Enalaprilat.

Burckhardt BB, Laeer S - Int J Anal Chem (2015)

Bottom Line: Enalapril, enalaprilat, and benazepril served as sample drugs.The applied sample preparation and extraction successfully reduced the absolute and relative matrix effect to comply with international guidelines.Recoveries ranged from 77 to 104% for enalapril and from 93 to 118% for enalaprilat.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-University, 40225 Düsseldorf, Germany.

ABSTRACT
In USA and Europe, medicines agencies force the development of child-appropriate medications and intend to increase the availability of information on the pediatric use. This asks for bioanalytical methods which are able to deal with small sample volumes as the trial-related blood lost is very restricted in children. Broadly used HPLC-MS/MS, being able to cope with small volumes, is susceptible to matrix effects. The latter restrains the precise drug quantification through, for example, causing signal suppression. Sophisticated sample preparation and purification utilizing solid-phase extraction was applied to reduce and control matrix effects. A scale-up from vacuum manifold to positive pressure manifold was conducted to meet the demands of high-throughput within a clinical setting. Faced challenges, advances, and experiences in solid-phase extraction are exemplarily presented on the basis of the bioanalytical method development and validation of low-volume samples (50 μL serum). Enalapril, enalaprilat, and benazepril served as sample drugs. The applied sample preparation and extraction successfully reduced the absolute and relative matrix effect to comply with international guidelines. Recoveries ranged from 77 to 104% for enalapril and from 93 to 118% for enalaprilat. The bioanalytical method comprising sample extraction by solid-phase extraction was fully validated according to FDA and EMA bioanalytical guidelines and was used in a Phase I study in 24 volunteers.

No MeSH data available.


Related in: MedlinePlus

Effect of elution volume on peak area of the analytes of interest. The peak areas of enalapril (a), enalaprilat (b), and benazepril (c) after elution are presented with different volumes of 2% formic acid in methanol. Each determination was conducted by three independently prepared quality control samples. The mean and corresponding standard deviations are shown.
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fig6: Effect of elution volume on peak area of the analytes of interest. The peak areas of enalapril (a), enalaprilat (b), and benazepril (c) after elution are presented with different volumes of 2% formic acid in methanol. Each determination was conducted by three independently prepared quality control samples. The mean and corresponding standard deviations are shown.

Mentions: Finally, the conducted investigations on the most appropriate elution solvent and its volume yielded 0.4 mL acidified methanol (2% formic acid, v/v). This elution step did not only served to elute the analytes of interest but also acted as a final step to fraction the analytes of interest and other interfering residual compounds. Hereby the choice and elution force of the solvent as well as the amount of solvent were investigated on their effect to attribute to a rugged, reliable, and selective protocol. As illustrated in Figure 6, there was a reciprocal relationship between the elution volume of acidified methanol and the detected peak areas of the analytes of interest. The obtained smaller peak areas—after the sorbent material was eluted with higher volumes of acidified methanol—might be explained by the fact that more interfering matrix was coeluted. The interference induced by the matrix led to ion suppression and smaller peak areas.


Sample Preparation and Extraction in Small Sample Volumes Suitable for Pediatric Clinical Studies: Challenges, Advances, and Experiences of a Bioanalytical HPLC-MS/MS Method Validation Using Enalapril and Enalaprilat.

Burckhardt BB, Laeer S - Int J Anal Chem (2015)

Effect of elution volume on peak area of the analytes of interest. The peak areas of enalapril (a), enalaprilat (b), and benazepril (c) after elution are presented with different volumes of 2% formic acid in methanol. Each determination was conducted by three independently prepared quality control samples. The mean and corresponding standard deviations are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4383429&req=5

fig6: Effect of elution volume on peak area of the analytes of interest. The peak areas of enalapril (a), enalaprilat (b), and benazepril (c) after elution are presented with different volumes of 2% formic acid in methanol. Each determination was conducted by three independently prepared quality control samples. The mean and corresponding standard deviations are shown.
Mentions: Finally, the conducted investigations on the most appropriate elution solvent and its volume yielded 0.4 mL acidified methanol (2% formic acid, v/v). This elution step did not only served to elute the analytes of interest but also acted as a final step to fraction the analytes of interest and other interfering residual compounds. Hereby the choice and elution force of the solvent as well as the amount of solvent were investigated on their effect to attribute to a rugged, reliable, and selective protocol. As illustrated in Figure 6, there was a reciprocal relationship between the elution volume of acidified methanol and the detected peak areas of the analytes of interest. The obtained smaller peak areas—after the sorbent material was eluted with higher volumes of acidified methanol—might be explained by the fact that more interfering matrix was coeluted. The interference induced by the matrix led to ion suppression and smaller peak areas.

Bottom Line: Enalapril, enalaprilat, and benazepril served as sample drugs.The applied sample preparation and extraction successfully reduced the absolute and relative matrix effect to comply with international guidelines.Recoveries ranged from 77 to 104% for enalapril and from 93 to 118% for enalaprilat.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-University, 40225 Düsseldorf, Germany.

ABSTRACT
In USA and Europe, medicines agencies force the development of child-appropriate medications and intend to increase the availability of information on the pediatric use. This asks for bioanalytical methods which are able to deal with small sample volumes as the trial-related blood lost is very restricted in children. Broadly used HPLC-MS/MS, being able to cope with small volumes, is susceptible to matrix effects. The latter restrains the precise drug quantification through, for example, causing signal suppression. Sophisticated sample preparation and purification utilizing solid-phase extraction was applied to reduce and control matrix effects. A scale-up from vacuum manifold to positive pressure manifold was conducted to meet the demands of high-throughput within a clinical setting. Faced challenges, advances, and experiences in solid-phase extraction are exemplarily presented on the basis of the bioanalytical method development and validation of low-volume samples (50 μL serum). Enalapril, enalaprilat, and benazepril served as sample drugs. The applied sample preparation and extraction successfully reduced the absolute and relative matrix effect to comply with international guidelines. Recoveries ranged from 77 to 104% for enalapril and from 93 to 118% for enalaprilat. The bioanalytical method comprising sample extraction by solid-phase extraction was fully validated according to FDA and EMA bioanalytical guidelines and was used in a Phase I study in 24 volunteers.

No MeSH data available.


Related in: MedlinePlus